Vol 2, Issue 3, 2014 ISSN: 2321-6832 Original Article USE OF AZOLLA BIOFERTILIZER IN POT CULTURE STUDIES WITH PADDY CROP ORYZA SATIVA MAHALINGAM.P.U, MUNIAPPAN.K*, ARUMUGAM.N AND SENTHIL MURUGAN.G ABSTRACT Department of Biology, Gandhigram Rural Institute Deemed University, Gandhigram, Dindigul District, Tamil Nadu Email: munistudy@gmail.com Received: 24 July 2014, Revised and Accepted: 14 August 2014 The pure cultures of Azolla species were mass multiplied and used as potential biofertilizer to study the growth performance of paddy (BPT paddy variety) in pot culture. Pots were prepared with different compositions of Red soil, Vermicompost, NPK and Azolla cultures. Healthy seeds of BPT paddy variety were collected from Raj Agro Form, Dindigul and showed in pots with thirteen treatments and watered regularly. The various growth parameters such as percentage of Seed germination, Shoot length, Root length, No of leaflets, No of root branches, Wet and Dry weight of whole crops of different treatment were observed periodically for 21 d. The total chlorophyll and carotenoid contents were estimated in all treatments including control. The rapid growth rate of paddy crop was recorded between 15 to 21 d of pot cultures due to the influence of Azolla biofertilizers. This study will attract the farmers to use Azolla biofertilizers for paddy cultivation. Keywords: Azolla, BPT Paddy, Oryza sativa, Biofertilizer. Nitrogen fixation INTRODUCTION The quick multiplication rate and rapid decomposing capacity of Azolla has become an important factor to used as green manure cum biofertilizer in paddy field [1]. This free-floating freshwater fern, fixes atmospheric nitrogen through the symbiotic association with Anabaena azollae that lives inside the dorsal lobes of Azolla leaves, potentially supplying a substantial amount of N2 to the paddy crop [2]. The genus of Azolla species is very sensitive to lack of water in aquatic ecosystems such as stagnant waters, ponds, ditches, canals or paddy fields. These areas may be seasonally covered by a mat of Azolla associated with other free-floating crops species such as Lemna minor [3]. Application of Azolla not only increases the growth and yield of paddy crops but also improves the soil fertility [4]. The mass cultivation of Azolla species is achieved in fresh water under strictly controlled conditions. The fresh water system is wildly available in populated countries like India; large scale production of Azolla is feasible in tropical conditions in developing countries, where land costs and labor are comparatively cheaper. The mass cultivation of Azolla in dairy effluent with various combinations of cow dung, whey water and tap water as low cost medium was reported [5]. Hence, utilization of waste water from dairy, industry also recommendable for mass cultivation of Azolla. This aquatic fern can be exploited as a potential source of bioferitilizer to increase the production of paddy variety [6]. Hence; Preliminary studies were carried out to evaluate the growth performance of paddy crop using Azolla biofertilizer. MATERIALS AND METHOD Mass multiplication of Azolla species The large cement tub was filled with 2 Kg of fine red soil. Slurry was made by mixing one Kg of cow dung, 15 gm of super phosphate in 5 liters of water. Only live and healthy culture of half kg Azolla was inoculated in the prepared tub bed. The optimum temperature 27 0 C and ph 5.0 to 5.7 is maintained for mass multiplication [7]. Population estimation of Azolla species Pure cultures of Azolla species were procured from TNAU, Coimbatore. The Population of Azolla was estimated by direct count method. Such as 50 gms wet wt of the culture was taken and spread over the marked 1 sq. cm area of the clean grid petriplate. Then it was observed under 50 lux white light source. The number of individual Azolla were counted per grid field and the populations were estimated by using Conn s direct observation method [8]. Pot culture studies with paddy crop using Azolla biofertilizer Pots were prepared with thirteen different treatment using red soil, vermicompost NPK and Azolla cultures. Healthy seeds of BPT paddy variety were collected from Raj Agro Farm dindigul and sowed in pots and watered regularly. The various growth parameters such as percentage of seed germination, Shoot length, Root length, No of leaflets, No of branches, Wet and Dry weight of whole crops of different treatment were observed periodically for 21 d. Analysis of Seed Germination Percentage of paddy crop The germination percentage of paddy seed in various treatments were calculated using the following Formula [9]. Germination (%) = Number of seeds germination / Total number of seeds X 100 Analysis of Shoot Length and Root Length of the paddy crop The shoot length of Azolla species treated paddy crops were measured in all 30 d treated crops. The average shoot length is expressed in centimeter for each treatment and in control. Simultaneously the length of root was measured from a fixed point below the surface of the soil to the end of the root [10]. Analysis of Fresh and Dry Weight of the paddy Crop The paddy crop from pots of each treatment were carefully uprooted on 15d & 21d, washed with water and weighted the fresh weight using weighing balance. Followed by the whole Crops were dried in hot air oven at 50 o C by gradually increasing the temperature up to 120 o C for every two hrs for 24hrs. The dry weight of whole crop was weighed accurately and the results were expressed in grams [11]. Estimation of Chlorophyll and Carotenoid Contents in Paddy Crop The chlorophyll & carotenoid contents of paddy crop grown in various treatments were estimated by standard method (Arnon, 1949). One gram of leaf from paddy crop was homogenized in 20 ml acetone (80%) and centrifuged at 10,000 rpm for 5 min. The contents of total chlorophyll (T-Chl), chlorophyll a (Chl-a), chlorophyll b (Chl-b) and carotenoid in the supernatant were determined by spectrophotometrically at OD of 645, 662 and 470
nm. The chlorophyll and carotenoid contents were calculated using the following formulae [12]: Chlorophyll a (mg/g fw) = 11.75*A662-2.35*A645 Chlorophyll b (mg/g fw) = 18.61*A645-3.96*A662 Total chlorophyll (mg/g fw) = Chlorophyll a + b Carotenoids (µmole g fw) =1000 X A470-2.29*chlorophyll (a) 81.4* chlorophyll (b) / 227. Statistical Analysis of Selected Parameters Statistical analysis of the data was carried out by using Prism Software (PV6). Mean standard deviation (SD) and percent variation was calculated. 'One Way Analysis of Variance' (ANOVA) was tested in order to check the statistical significance in parameters like Root and Shoot length, No of leaflets, No of root branches, Fresh and Dry wt of treated paddy crops. The data was analyzed for three different levels of significance based on the p values RESULT AND DISCUSSION Azolla is a free floating aquatic fern, which can be widely found in freshwater environments in temperate and tropical regions all over the world. It ranks among the fastest growing crops on earth and due to its association with the nitrogen fixing Cyanobacteria Anabaena azollae, it is independent of external organic nitrogen. In that way Azolla species is not only known to fix huge amounts of carbon, but as well to produce vast amounts of organic nitrogen. The nutrient, which mainly limits the growth of Azolla, is phosphorous. Azolla comes under the family of Salviniaceae [13].In the present study the procured strains of Azolla were mass multiplied under controlled condition (FIG 1) without Lemna species contamination. The dead Azolla cultures were removed periodically in order to enhance the oxygen content for a time. The well grown Azolla cultures have been treated with the selected paddy cultivars for 21 days. Fig. 1: Mass multiplied Azolla Species Seed germination is the growth of an embryonic crop contained within a seed; it results in the formation of the seedling, in the present study the pots were prepared in different combinations of red soil, vermicompost, NPK and Azolla. The sowed paddy seeds germination percentage was observed with in 7 to 10 d under controlled conditions. Approximately 95% seeds are germinated well compared with control pot (TABLE 1). The growth of shoot length on 15d & 21d was observed as 8.9±1.05 & 16.83±0.351 cm in treatment 12, this drastic growth may be the influence of Azolla. The graphical comparison of shoot length on 15 th and 21 st d given in (FIG 2) Fig. 2: Root and shoot length of paddy cultivars grown in various treatments on 15d and 21d. Well development of leaflets in (Treatment 12) and root branches T12 were observed in the Azolla treated paddy crops during the same period of course. The more number of leaflets may enhance the rate of photosynthesis which helps to produce more number of carbohydrate productions in the form of reserved food materials in seeds due to presence of increased level of chlorophyll contents [14] The enhanced level of chlorophyll content in the treated crops was higher compared to control (FIG 2 and TABLE 4), which was observed in the treatment pots 1, 7 and 12 has high level of chlorophyll content. The mean value of total chlorophyll content was expressed in mg/g in TABLE: 4. the enormous development of roots in the crops plays a major role in absorption of minerals and available nutrients in the soil under controlled condition [15] (TABLE 1). Comparatively increased level of wet and dry weight was observed in 15 th and 21 st d of paddy cultivars of Azolla treated crops. (TABLE 3) This indirectly indicates the enhancement of Biomass level [16].The statistical analysis of selected parameter (TABLE 5) showed statistically significant result due to the impact of Azolla in the present study. Table 1: Percentage of seed Germination of paddy Oriza sativa in various treatments on 7d Treatment Treatment Condition No of seeds Germination % of germination T0 Sand + Red sail (1:1) - Control T1 Recommended dose NPK (100%) T2 Vermicompost (100%) T3 Azolla microphylla (100%) T4 Azolla filiculoids C4 hybrids (100%) T5 Azolla microphylla + Azolla filiculoids C4 hybrids T6 14.66 1.52 18.33 0.57 17.66 18.66 0.57 73.33 91.67 88.33 93.33 2
Azolla microphylla + NPK T7 C4 hybrids + NPK T8 Azolla microphylla (25%) + NPK T9 Azolla microphylla + vermicompost T10 Sand+ Red soil(1:1) + (C4 hybrids + vermin compost T11 Sand+ Red soil (1:1) + Azolla microphylla (25%) + Vermicompost 15.66 1.52 18 1.73 19.33 0.57 18.66 0.57 78.33 88.33 96.67 95.33 T12 Azolla microphylla (25%) vermicompost (25% ) + NPK (25% ) Values are mean of triplicate ± standard error 19 0 Table 2: Number of leaf lets and Root branches in paddy crop on 15 d and 21 d 95.00 Treatment Treatment Condition Number of leaf lets Number of root branches (in cm) 15 d 21 d 15 d 21 d T0 Sand + Red sail (1:1) - Control 1 2 4.66 0.57 5.0 1.0 T1 Recommended dose NPK (100%) T2 Vermicompost (100%) T3 Azolla microphylla (100%) T4 Azolla filiculoids C4 hybrids (100%) T5 Azolla microphylla + Azolla filiculoids C4 hybrids T6 Azolla microphylla + NPK T7 C4 hybrids + NPK T8 Azolla microphylla (25%) + NPK T9 Azolla microphylla + vermicompost T10 Sand+ Red soil(1:1) + (C4 hybrids + vermin compost T11 Sand+ Red soil (1:1) + Azolla microphylla (25%) + Vermicompost 3 4 3 4 4.66 1.52 8.0 1.73 4.0 1.73 6.33 1.52 5.0 1.0 6.66 0.57 3.33 0.57 6.0 2.0 5.0 1.0 6.0 1.0 3.66 2.08 10.0 1.0 3.0 1.0 8.66 1.52 4.33 1.15 7.66 1.52 5.33 0.57 6.33 0.57 4.66 1.52 5.33 1.15 4.66 1.52 6.66 0.57 T12 Azolla microphylla (25%) Azolla filiculoids C4 hybrids (25%) +vermicompost (25% ) + NPK (25% ) 3.66 2.08 10.0 1.0 Value are mean of triplicate ± standard error 3
Table 3: Fresh weight and Dry weight of whole paddy crop on 15 d and 21 d Treatment Treatment Condition Fresh weight in whole paddy crop(in mg) Dry weight in whole paddy crop(in mg) 15 d 21 d 15 d 21 d T0 Sand + Red sail (1:1) - Control 0.02 0.01 0.022 0.04 0.008 ± 0.002 0.011±0.0017 T1 Recommended dose NPK (100%) T2 Vermicompost (100%) T3 Azolla microphylla (100%) T4 Azolla filiculoids C4 hybrids (100%) T5 Azolla microphylla + Azolla filiculoids C4 hybrids T6 Azolla microphylla + NPK T7 C4 hybrids + NPK T8 Azolla microphylla (25%) + NPK T9 Azolla microphylla + vermicompost T10 Sand+ Red soil(1:1) + (C4 hybrids + vermin compost T11 Sand+ Red soil (1:1) + Azolla microphylla (25%) + Vermicompost 0.02 0.04 0.043 0.001 0.0093 ±0.0032 0.027 ±0.002 0.04 0.01 0.050 0.031 0.014 ±0.0025 0.029 ± 0.021 0.03 0.008 0.044 0.012 0.028 ±0.005 0.038 ±0.044 0.033 0.006 0.038 0.003 0.008 ±0.0028 0.024 ±0.004 0.033 0.005 0.041 0.0005 0.009 ±0.0046 0.082 ±0.010 0.045 0.013 0.041 0.014 0.008 ±0.0026 0.023 ±0.005 0.03 0.02 0.045 0.0050 0.009 ±0.0020 0.027 ±0.005 0.03 0.09 0.035 0.008 0.007 ±0.0045 0.017 ±0.009 0.04 0.04 0.037 0.005 0.008 ±0.001 0.030 ±0.004 0.04 0.02 0.045 0.005 0.011 ±0.015 0.016 ±0.004 0.03 0.01 0.040 0.001 0.008 ±0.0047 0.012 ±0.005 T12 Azolla microphylla (25%) Azolla filiculoids C4 hybrids (25%) +vermicompost (25% ) + NPK (25% ) 0.03 0.007 0.051 0.008 0.006 ±0.0015 0.01 ±0.007 Value of mean triplicate ± standard error Table 4: Chlorophyll and Carotenoid contents of paddy crop grown in various treatmentson 21d Chlorophyll a Total Treatment Treatment condition (mg/g fw) Chlorophyll b Chlorophyll (mg/g fw) Carotinoids (mg/g fw) (μ mole g /fw) T0 Sand + Red sail (1:1) Control T1 Recommended dose NPK (100%) 4.50±0.636 2.242±0.253 6.74 1.68±0.044 10.08±1.732 1.210±0.590 11.29 3.89±0.015 T2 Vermicompost (100%) 5.06±0.999 2.820±1.04 7.88 1.46±0.04 T3 Azolla microphylla (100%) 5.17±0.61.305±0.932 7.475 1.19±0.125 T4 Azolla filiculoids C4 hybrids (100%) T5 Azolla microphylla + 4.48±0.670 2.89±0.60 7.37 1.55±0.020 5.15±1.558 2.905±1.254 8.05 2.18±0.025 4
Azolla filiculoids C4 hybrids T6 Azolla microphylla + NPK T7 C4 hybrids + NPK T8 Azolla microphylla (25%) + NPK T9 Azolla microphylla + vermicompost Sand+ Red soil(1:1) + T10 7.57±1.515 2.762±1.172 10.33.75±0.043 6.91±2.035 3.265±0.70 10.175 2.93±0.02 8.34±0.57 2.19±1.28 10.53 2.946±0.037 7.55±0.023 2.976±0.96 10.526 2.56±0.01 (C4 hybrids + vermin compost Sand+ Red soil (1:1) + T11 Azolla microphylla (25%) + Vermicompost T12 Azolla microphylla (25%) Azolla filiculoids C4 hybrids (25%) +vermicompost (25% ) + NPK (25% ) 8.63±0.23 1.218±0.245 9.848 3.106±0.032 7.15±0.23 3.601±0.93 10.751 2.033±0.028 8.32±0.576 3.042±0.894 11.36.166±0.015 Value of mean triplicate ± standard error Table 5: Statistical analysis on different growth parameters of paddy cultivars growth in various treatments Parameters 15 th d treatment p value Level of significance With mean 21 st d treatment p value Level of significance With mean Root Length in cm 0.0269 *4.573 ± 0.4818 < 0.0001 ****6.372 ± 0.3524 Shoot Length in cm < 0.0001 ****9.517 ± 0.2485 < 0.0001 ****14.75 ± 0.3582 No of Leaflets < 0.0001 ****2.552 ± 0.08535 <0.0004 ***2.083 ± 0.05921 No of Root branches in cm < 0.0001 ****7.024 ± 0.4968 < 0.0001 ****7.303 ± 0.4524 Fresh wt of whole crop in mg < 0.0001 ****0.0340 ± 0.002026 < 0.0001 ****0.0425 ± 0.001406 Dry wt of whole crop in mg 0.1653 Not significant 0.0051 **0.02792 ± 0.005442 Significant (p = 0.01 to 0.05), ** Very Significant (p = 0.001 to 0.01) and *** Extremely Significant (p < 0.001) **** strongly significant (p<0.0001) CONCLUSION In the present preliminarily study, the procured Azolla species were mass multiplied in controlled condition and the same was utilized for the growth of paddy crops for 30 d. During the course of studies for 30 d the fundamental parameters were investigated. It was observed that, Azolla species started to show their impact from the 15 th d and was completely degraded in the prepared tub bed on the 21 st d. Logarithmic growth rate of paddy crop was observed between 15 th to 21 st d of treatment. Hence further supplementation is recommended to enhance the growth and yield of paddy crops for longer time till harvesting. The functional role and interaction between nitrogen fixation and utilization by paddy crop networks per square feet area namely measurement of Azolla response, grain yield, Crop Nitrogen uptake, excretion of NH3 by Azolla species in free state conditions, rate of reproduction through sporulation, preparation of dried inoculum along with long shelf life of different Azolla species are yet to be analyzed well. An extensive research work has to be carried out to produce more quintals of paddy yield using organic fertilizers, which will help to increase the economic status of our rural parts of India. ACKNOWLEDGMENT Authors are grateful to TNAU for providing the various cultures of Azolla species and Raj agro farms Dindigul, for the supply of BPT paddy seeds. AUTHOR S CONTRIBUTION MPU, MK and SMG designed the experimental setup. MK, SMG carried out the implementation of various methods. MPU, AN interpreted the results and AN prepared the manuscript. All authors read and approved the manuscript. 5
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